SUPPORTING INFORMATION
For
PHYTOESTROGENS IN THE ENVIRONMENT: II. MICROBIOLOGICAL
DEGRADATION OF PHYTOESTROGENS AND THE RESPONSE OF FATHEAD
MINNOWS TO DEGRADATE EXPOSURE
MEGAN M. KELLY†, NATHAN T. FLEISCHHACKER‡, DANIEL C. REARICK§, WILLIAM A.
ARNOLD†‡, HEIKO L. SCHOENFUSS§, AND PAIGE J. NOVAK†‡*
† Water Resources Science Graduate Program, University of Minnesota, St. Paul,
Minnesota, USA
‡ Department of Civil Engineering, University of Minnesota, St. Paul, Minnesota,
USA
§ Aquatic Toxicology Laboratory, St. Cloud State University, St. Cloud,
Minnesota, USA
1
METHODS
Table S1. Nitrification reactor media
Concentration
Chemical
Formula
mg/L
Sodium Phosphate
Na2HPO4-7H2O
Potassium
KH2PO4
3,000
83.3
Phosphate
Magnesium Sulphate
MgSO4-7H20
80
Calcium Chloride
CaCl2
75
Sodium Bicarbonate
NaHCO3
1.5
Ferric Chloride
FeCl3-6H2O
0.8
Copper Sulphate
CuSO4
0.2
EDTA
Na3EDTA-4H2O
1
Cobalt Chloride
CoCl2-6H2O
2.0 x 10-4
Zinc Sulphate
ZnSO4-7H2O
0.1
Sodium Molybdate
Na2MoO4-2H2O
0.1
Manganese Chloride
MnCl2-2H2O
2
Ammonium Sulphate
(NH4)2SO4
1,000
2
Table S2. Confirmatory water chemistry for the larval minnow phytoestrogen exposure
experiment (mean ± st. err.; n=2) averaged over the exposure period.
Treatment
Genistein
Daidzein
Formononetin
Lake Water Control
Non-detect
40 ± 57 ng/L
25 ± 35 ng/L
Degraded genistein
30 ± 42 ng/L
115 ± 163 ng/L
Non-detect
Degraded daidzein
Non-detect
140 ± 198 ng/L
30 ± 42 ng/L
Degraded mixture
70 ± 0 ng/L
90 ± 127 ng/L
Non-detect
Note: the daidzein detection in all the treatments was thought to be a co-eluting
compound as a result of biological activity in the system. A similar compound was
detected in similar studies fed parent (non-degraded) phytoestrogens [S1]. In addition,
similar studies showed that daidzein exposure did not cause a measureable response
larval minnows [S1]; therefore, the presence of either daidzein (unlikely) or a co-eluting
compound in these samples (likely) was deemed to be unimportant with respect to
larval response.
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Table S3. Concentrations of phytoestrogens measured (mean ± standard deviation;
n=5) in the ethanol blank [further diluted with well water 1:588,000 prior to minnow
exposure], the bioreactor feed, and in the bioreactor effluent [further diluted with well
water 1:400 prior to minnow exposure] in the experiment to test the effect of adult
minnow exposure to the biodegraded phytoestrogens.
Treatment
Ethanol blank
Bioreactor feed
Genistein
Daidzein
Formononetin
17.6 ± 2.4 ng/L
Non-detect
8.8 ± 0.7 ng/L
108,810 ± 8,210 ng/L
65,260 ± 5,640 ng/L
163,050 ± 6,620 ng/L
34.3 ± 33.6 ng/L
Non-detect
8.5 ± 0.8 ng/L
Bioreactor
effluent
Optical density (OD), suspended solids (SS), volatile suspended solids (VSS),
and dissolved organic carbon (DOC) analysis. OD was measured using a Beckman DU
530 UV/VIS Spectrophotometer (Fullerton, CA) at a wavelength of 600 nm. Well-mixed
samples (2 mL) were placed in cuvettes (Life Sciences, Foster City, CA) and measured
three times; the average value was recorded. SS and VSS were measured according to
Standard Method 2540D and 2540E [S2], respectively. Samples (20 mL) were analyzed
for DOC by filtering them through a GFF, acidifying the filtrate to pH 2 with 5 M H 2SO4,
purging inorganic carbon with N2 gas, then analyzing the residual carbon (assumed to
be organic) with a Sievers 900 Portable TOC Analyzer (General Electric, Fairfield, CT).
4
Ion analysis. Nitrate and nitrite concentrations were determined using a Metrohm
(Riverview, FL, USA) 761 ion chromatograph using a Metrohm 766 sample processor
and IC Net software. The eluent solution consisted of 1mM NaHCO3 and 32 mM
Na2CO3. Regenerant was a 0.2 mM sulfuric acid solution. A combined external
calibration curve for nitrate and nitrite in Milli-Q was used to quantify nitrate and nitrite.
5
RESULTS
120
Concentration (g/L)
100
80
60
40
20
0
0
10
20
30
50
40
60
Time (hrs)
Figure S1. Degradation of genistein (red) and daidzein (blue) in Mississippi River water
collected on June 26, 2013, incubated at 20° C. Long-dash lines represent fits to the
Gompertz equation, short-dash lines to zero-order kinetics, and dash-dot lines to firstorder kinetics. Empty circles represent controls.
6
120
A
Concentration (g/L
100
10
B
8
80
6
60
4
40
2
20
0
0
2.0
C
0.8
D
1.8
Concentration (g/L
1.6
0.6
1.4
1.2
1.0
0.4
0.8
0.6
0.2
0.4
0.2
0.0
0
20
40
60
80
0.0
100
Time (hrs)
0
20
40
60
80
100
Time (hrs)
Figure S2. Degradation of genistein with an initial concentration of 50 μg/L (A), 10 μg/L
(B), 1 μg/L (C), and 0.5 μg/L (D) in Mississippi River water collected on June 26, 2013,
incubated at 20° C. Long-dash lines represent fits to the Gompertz equation, short-dash
lines to zero-order kinetics, and dash-dot lines to first-order kinetics. Empty circles
represent controls.
7
70
12
A
Concentration (g/L)
60
10
50
8
40
6
30
4
20
2
10
0
0
1.4
0.6
C
1.2
Concentration (g/L)
B
D
0.5
1.0
0.4
0.8
0.3
0.6
0.2
0.4
0.2
0.1
0.0
0.0
0
20
40
60
80
100
Time (hrs)
0
20
40
60
80
100
Time (hrs)
Figure S3. Degradation of daidzein with an initial concentration of 50 μg/L (A), 10 μg/L
(B), 1 μg/L (C), and 0.5 μg/L (D) in Mississippi River water collected on June 26, 2013,
incubated at 20° C. Long-dash lines represent fits to the Gompertz equation, short-dash
lines to zero-order kinetics, and dash-dot lines to first-order kinetics. Empty circles
represent controls.
8
50
Inhibitor added
40
mg N/L
30
20
10
0
0
10
20
30
40
50
60
Time (hrs)
Figure S4. Concentrations of nitrite (black), nitrate (white), and ammonia (grey) in an
enriched culture of nitrifying organisms before and after the addition of allylthiourea.
9
Figure S5. Percent larval fathead minnow survival during a 21-day exposure to the
degradation compounds singly and in mixture. Initial cohorts were established using 75
larvae per treatment. Survival represents number of individuals remaining after
behavioral testing.
10
Figure S6. Larval fathead minnow escape performance following 21-day exposure to
degraded phytoestrogen compounds. C-start escape performance was quantified using.
(A) body length (mm); (B) mean latency (ms) from stimulus to response; (C) mean
escape velocity relative to body length (BL/ms); (D) mean total escape performance
(BL/ms) defined as (distance travelled/BL)/(40+latency) (n = 30 for all treatments).
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Figure S7. Mean ± standard error plasma vitellogenin concentrations (g/mL) in male (A)
and female (B) fathead minnows exposed for 21- days to the degraded phytoestrogen
mixture (Daidzein, Genistein, and Formononetin 1,000 ng/L per compound). Severity of
hepatocyte vacuole presence for male (C) and female (D) minnows. Testis (E) and ovarian
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(F) maturity. Sample size for each treatment is listed in each column. Sample sizes differed
between endpoints and treatments due to insufficient tissue or analysis failure.
13
Figure S8. Body condition factor (A-male; B-female), hepatosomatic idnex (C,D) and
gonadosomatic index (E,F) for fathead minnows exposed for 21 days to the degradation
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mixture. Sample size for each treatment (and all three endpoints) is listed in each column
of (A) and (B), respectively.
15
Figure S9. Mean ± standard error expression of secondary sex characteristics (A) and
total aggression index (B) for male fathead minnows exposed 21-days to the
degradation mixture. Sample size for each treatment is listed in each column
REFERENCES
S1. Rearick DC, Fleischhacker NT, Kelly MM, Arnold, WA, Novak PJ, Schoenfuss HL.
Environmental occurrence of phytoestrogens and exposure effects on fathead
minnows. Environ. Toxicol. Chem. submitted.
S2. Yi T, Harper W. 2007. The link between nitrification and biotransformation of 17αethinylestradiol. Environ. Sci. Technol. 41:4311-6.
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